Introgression Pathway for Drought Tolerance In
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Texas A&M Repository INTROGRESSION PATHWAY FOR DROUGHT TOLERANCE IN PEANUT (Arachis hypogaea L.) A Dissertation by JOHN MICHAEL CASON Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Charles E. Simpson Co-Chair of Committee William L. Rooney Committee Members, Jason E. Woodward Peter A. Dotray Head of Department, David D. Baltensperger December 2018 Major Subject: Plant Breeding Copyright 2018 John Michael Cason ABSTRACT In this study, a hybrid of the bridge species Arachis vallsii Krapov. and W.C. Greg. (VSW 9902-1) and A. dardani Krapov. and W.C. Greg. (GK12946) was created to initiate an introgression pathway for movement of possible drought tolerance genes into the cultivated peanut (A. hypogaea L.). A hybrid between the two species was successfully created and confirmed based on leaf morphology, pollen counts and intermediated leaf morphology. One-hundred and seventy-five attempts were made to double the chromosome complement using 3 methods at concentrations of 0.02% and 0.03% colchicine for exposure times ranging from 6 to 24 hours. No attempt has been successful to date. In addition, a greenhouse transcriptome study with 7 day-imposed drought was conducted on A. dardani (12946) and the reference species A. ipaënsis (Krapov. and W.C. Greg.) (KGBPScS-30076) (B genome donor of the cultivated peanut). Differential gene expression analysis (EdgeR Test) of the normalized RPKM (Reads Per Kilobase Million mapped reads) values was conducted with a fold value > abs (2) at the p ≤ 0.05 level using CLC Genomics Workbench v8. Significant transcript levels associated with drought tolerance were found in relation to the putative drought species (A. dardani (12946)), which have not been reported previously. Transcripts were identified that were higher between physiological states and between species. In total, 40 genes were identified for further study. ii ACKNOWLEDGEMENTS I would like to take this opportunity to thank those who have helped me during my time in graduate school. First and foremost, I would like to thank my Lord and Savior Jesus Christ for saving me and giving me this opportunity. Next, I would like to thank Dr. Charles Simpson, he has been an invaluable mentor both academically and professionally, as well as providing me the means to attend graduate school. His unique experiences traveling South America have given him knowledge about the genus Arachis that has been invaluable to me in my research. In addition, his unwavering support has been very much appreciated. I would also like to thank Mr. Michael Baring for his listening ear and sound advice. I have spent countless hours traveling the state of Texas with him asking questions and honing the skills that I have learned in the classroom. I would also like to thank numerous others. The other members of my committee. Dr. Bill Rooney for guidance in academic matters and advice, Drs. Jason Woodward and Peter Dotray for their encouragement and support, LeAnn Hague for being my eyes and ears on campus and Dr. Jeff Brady, Brian Bennett, Nichole Cherry and Chase Murphy for all the assistance in my research. I would also like the thank Dr. Craig Nessler, Dr. David Baltensperger and Dr. Don Cawthon for their support of my education. Last but certainly first in my heart is my wife, Nicole Cason. She has had to listen to me complain and stress out over deadlines. She has been a constant source of encouragement through the long hours of studying. She has taken up the slack for me in order to keep our family going and I could not have done this without her. iii CONTRIBUTORS AND FUNDING SOURCES Funding for education costs and research was supported by personal contributions by Dr. Charles E. Simpson, internal Texas A&M AgriLife Research funds made available by Dr. Craig L. Nessler, Dr. David D. Baltensperger and Dr. Donald L. Cawthon. In addition, a Texas A&M Genomics seed grant was used to conduct part of the transcriptomics study. This work was supported by a dissertation committee consisting of Dr. Charles E. Simpson (research advisor/co-chair) and Dr. William L. Rooney (academic advisor/co-chair), Dr. Peter A. Dotray of the Department of Soil and Crop Sciences and Dr. Jason E. Woodward of the Department of Plant Pathology and Microbiology. The analyses depicted in Chapter IV were advised by Dr. Jeffery A. Brady. In addition, Brian Bennett, Nichole Cherry and Chase Murphy assisted in data collection. iv TABLE OF CONTENTS Page ABSTRACT ......................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................ iii CONTRIBUTORS AND FUNDING SOURCES ................................................................iv TABLE OF CONTENTS ...................................................................................................... v LIST OF FIGURES ............................................................................................................ vii LIST OF TABLES ............................................................................................................ viii CHAPTER I INTRODUCTION ........................................................................................... 1 CHAPTER II LITERATURE REVIEW ............................................................................... 5 II.1 Taxonomy and Organization of Genus Arachis and its Species ................................. 5 II.2 Drought Tolerance ....................................................................................................... 8 II.3 Breeding Strategies .................................................................................................... 11 II.4 Genomics and Molecular Markers............................................................................. 13 II.5 Chromosome Doubling Compounds ......................................................................... 16 II.6 Gene Introgression ..................................................................................................... 17 CHAPTER III MATERIALS AND METHODS ................................................................ 21 III.1 RNA-seq ................................................................................................................... 21 III.1.1 Greenhouse Study .............................................................................................. 21 III.1.2 RNA Extraction and Sequencing ....................................................................... 24 III.2 Crossing .................................................................................................................... 26 CHAPTER IV RESULTS AND DISCUSSION ................................................................. 32 IV.1 Relative Water Content ............................................................................................ 32 IV.2 Differential Gene Expression Analysis .................................................................... 34 IV.3 Crossing ................................................................................................................... 48 CHAPTER V CONCLUSIONS .......................................................................................... 55 REFERENCES .................................................................................................................... 58 v Page APPENDIX ........................................................................................................................ 75 vi LIST OF FIGURES Page Figure 1. A picture showing the crossing block layout with an A. vallsii female plant in a 36.2 cm basket with marked pollinations and hybridization isolation pots ........... 27 Figure 2. A picture showing the difference in the root systems of A. dardani and A. ipaënsis at 75 days after planting (DAP)....................................................................... 33 Figure 3. A picture documenting the presence of plant hairs and leaf angle adjustment in A. dardani. .............................................................................................. 34 Figure 4. A figure showing 8 shoot tissue DGE comparisons and the number of genes significantly up or down regulated 2 fold at an FDR-corrected p-value ≤ .05. ............. 36 Figure 5. A figure showing 8 root tissue DGE comparisons and the number of genes significantly up or down regulated 2 fold at an FDR-corrected p-value ≤.05. .............. 37 Figure 6. A figure depicting various transcription factors and their role in drought response (reprinted from Lata and Prada, 2011). ......................................................... 44 Figure 7. Pictures contrasting the leaf morphlogy of (clockwise): a. A. vallsii, b. A. dardani as compared to the intermediate morphlogy of c. A. vallsii x A. dardani hybrid and the flower morphology of the hybrid .......................................................... 51 Figure 8. A picture of an hybrid seed following colchicine treatment that is showing some promise of chromosme doubling ......................................................................... 53 vii LIST OF TABLES Page Table 1. A table showing crossing block information with the male and female parent, planting dates, first flower dates and